Conformational Changes in the BSA-LT4 Complex Induced by the Presence of Vitamins: Spectroscopic Approach and Molecular Docking.

Levothyroxine (LT4) is known for its use in various conditions including hypothyroidism. LT4 interaction with serum albumin may be influenced by the presence of vitamins. For this reason, we investigated the effect of vitamin C, vitamin B12, and folic acid on the complex of Bovine Serum Albumin with LT4 (BSA-LT4). UV-Vis spectroscopy was used to monitor the influence of vitamins on the BSA-LT4 complex. Fluorescence spectroscopy revealed a static quenching mechanism of the fluorescence of BSA-LT4 complex by the vitamin C and folic acid and a combined mechanism for vitamin B12. The interaction of vitamin C and folic acid with BSA-LT4 was moderate, while the binding of vitamin B12 was much stronger, extending the storage time of LT4 in blood plasma. Synchronous fluorescence found that the vitamins were closer to the vicinity of Trp than to Tyr and the effect was more pronounced for the binding of vitamin B12. The thermal stability of the BSA-LT4 complex was more evident, but no influence on the stability of BSA-LT4 complex was obtained for vitamin C. Molecular docking studies showed that vitamin C and folic acid bound the same site of the protein, while vitamin B12 bonded to a different site.

Evaluation of the interaction of levothyroxine with bovine serum albumin using spectroscopic and molecular docking studies.

Bovine serum albumin (BSA) acts as a carrier for many endogenous and exogenous compounds, such as thyroid hormones or corresponding drugs. Binding of the hydrophilic levothyroxine drug (LT4) to BSA is of significant pharmacological importance. In this work, UV-vis measurements were used to determine the pH value at which LT4 interacts optimally with proteins. The binding mechanism and affinity of the interaction between LT4 and BSA were investigated using Fourier-transform infrared spectroscopy (FT-IR), fluorescence, fluorescence resonance energy transfer (FRET), Surface Plasmon Resonance (SPR), supplemented by molecular docking analysis. Fluorescence measurements revealed the quenching effect of LT4 on the BSA intrinsic fluorescence and LT4 binding with BSA is driven by a ground-state complex formation that may be accompanied by a nonradiative energy transfer process. The thermodynamic parameters correspond to an enthalpic process, driven mainly by hydrogen bonds and van der Waals forces. Using SPR, the adsorbed amount of biomolecules was calculated and the binding affinity of LT4 with confined-BSA was characterized, indicating that the BSA immobilization plays an important role in LT4 binding. Docking studies confirmed the formation of the LT4-BSA complex with LT4 bound to site I on the BSA structure mainly with amino acid residues Trp 213, Tyr 137, Tyr 147. The calculation of the apparent association constant confirms the result obtained in SPR.Communicated by Ramaswamy H. Sarma.

Insight into dual fluorescence effects induced by molecular aggregation occurring in membrane model systems containing 1,3,4-thiadiazole derivatives.

This work reports on biophysical insights into the excited state intramolecular proton transfer (ESIPT) processes taking place in three 1,3,4-thiadiazole derivatives that served as model compounds, on which electronic absorption, fluorescence, Fourier-transform infrared spectroscopy (FTIR), surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies were performed. The fluorescence spectra recorded in various solvents revealed an interesting dual fluorescence effect. In molecules in their monomeric form, the effect is associated with the ESIPT phenomenon, and may be further enhanced by aggregation-related effects, such as aggregation-induced emissions. Other spectroscopic studies on the selected molecules in a liposomal medium as a model revealed that, in a biomimetic environment, they can exist in both monomeric and aggregated forms. In both cases, however, the effects observed are closely related to the lipid's main phase transition temperature and the structure of the molecule. Introduction of specific substituents to the phenyl moiety either allows or prevents proton transfer from occurring in the excited state. The hydrophobicity changes in a lipid environment may result in an emergence of specific molecular forms and therefore either facilitate or hinder ESIPT processes. SPR and EIS confirmed the significant hydrophobicity changes in the model lipid systems, while FTIR measurements revealed a notable influence of 1,3,4-thiadiazoles on the fluidity of liposomal membranes. The results obtained clearly show that the thiadiazole derivatives are very good model molecules for studying hydrophobic-hydrophilic environments, and particularly with polymers or liposomes used as drug delivery systems.

Electrochemical synthesis and characterization of poly(thionine)-deep eutectic solvent/carbon nanotube-modified electrodes and application to electrochemical sensing.

Electropolymerization of thionine (TH) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrodes (GCE) in ethaline deep eutectic solvent (DES) was carried out for the first time, to prepare poly(thionine) (PTH) films with different nanostructured morphologies. PTH films were formed on MWCNT/GCE by potential cycling electropolymerization in ethaline with the addition of different acid dopants CH3COOH, HClO4, HNO3, H2SO4 and HCl, acetic acid being the best. The electropolymerization process was monitored with an electrochemical quartz crystal microbalance. The polymerization scan rate was a key factor affecting the electrochemical and morphological properties of the PTHEthaline-CH3COOH/MWCNT/GCE; electrodeposition at 200 mV s-1 showing the best performance. The PTH/MWCNT/GCE platform was characterized using cyclic and differential pulse voltammetry, electrochemical impedance spectroscopy and scanning electron microscopy. The analytical characteristics of the PTH films were evaluated for sensing of ascorbic acid and biosensing of uric acid. The developed sensor exhibited a low detection limit (1.1 µM), wide linear range (2.8-3010 µM) and high sensitivity (1134 µA cm-2 mM-1) for ascorbic acid. After immobilization of uricase, UOx, on PTH/MWCNT/GCE, the biosensor was successfully applied to the determination of uric acid, with fast response (˂ 7 s), good sensitivity (450 µA cm-2 mM-1, wide linear range (0.48-279 µM) and low detection limit (58.9 nM), better than in the literature and than with PTH prepared in aqueous solution. The determination of uric acid in synthetic urine samples was successfully tested and the mean analytical recovery was 100.8 ± 1.4%. This is a promising approach for the determination of uric acid in real samples. Graphical abstract.

Biosensors for Antioxidants Detection: Trends and Perspectives.

Herein we review the recent advances in biosensors for antioxidants detection underlying principles particularly emphasizing advantages along with limitations regarding the ability to discriminate between the specific antioxidant or total content. Recent advances in both direct detection of antioxidants, but also on indirect detection, measuring the induced damage on DNA-based biosensors are critically analysed. Additionally, latest developments on (bio)electronic tongues are also presented.

Monitoring biomolecular interaction between folic acid and bovine serum albumin.

Folic acid is a bioactive food component whose deficiency can lead to a variety of health problems, while a high intake of folic acid can reduce the cytotoxicity of natural killer cells. The binding mechanism of folic acid to free bovine serum albumin (BSA) was studied using fluorescence, while the biomolecular interaction between confined-BSA and free folic acid was assessed by electrochemical methods and surface plasmon resonance. The fluorescence quenching mechanism of BSA by folic acid was found to have a static character. The thermodynamic parameters of the interaction were determined and indicated a spontaneous exothermic process with a binding constant of 8.72 × 104 M-1 at 25 °C. Confinement of BSA to gold surfaces occurred through different immobilization methods (static and hydrodynamic), inducing conformational changes, which influenced the orientation of BSA molecules binding sites towards free folic acid. The apparent binding constant using electrochemical methods (voltammetry and impedance spectroscopy) was only 5 times higher (41 and 37 × 104 M-1) compared to BSA free in solution, while for surface plasmon resonance, where the hydrodynamic immobilization method was used, the value was much higher (19 × 106 M-1). This work gives also an insight on the interaction of BSA with gold substrates, surface plasmon resonance enabling the calculation of the adsorbed amount. The obtained results help understanding the specific interaction between free and confined BSA with free folic acid.

Bioelectrochemical evaluation of plant extracts and gold nanozyme-based sensors for total antioxidant capacity determination.

The antioxidant properties of different plant extracts are usually claimed and used by food, medicine and cosmetic industry due to their health promoting capacities. In this study the presence of antioxidant compounds and the total antioxidant capacity of water-soluble extracts, prepared using two extraction methods and a variety of solvents, have been determined and a rapid screening method has been developed. Plant extracts characterisation, composition and antioxidant properties were confirmed by FTIR and Raman spectroscopies. Voltammetry, amperometry and electrochemical impedance were used to highlight the total antioxidant capacity of each extract using an electrochemical gold nanozyme-sensor based on the enzyme-like catalytic activity of gold nanoparticles. Both anodic area of cyclic voltammograms (10.31 µA V) and electrochemical index (153) calculated using differential potential voltammetry show the total content of antioxidant compounds, allowing to discriminate between different extracts. Amperometric total antioxidant capacity measurements were associated with those from classical chemiluminescence and good correlation has been found (Pearson's correlation coefficient of 0.958).

A Nanoparticle-Based Label-Free Sensor for Screening the Relative Antioxidant Capacity of Hydrosoluble Plant Extracts.

One of the most important aspects of the detection of antioxidant compounds is developing a fast screening method. The screening of the overall relative antioxidant capacity (RAC) of several Romanian hydrosoluble plant extracts is the focus of this work. This is important because of the presence of increasing levels of reactive oxygen species (such as H2O2) generates oxidative stress in the human body. The consequences are a large number of medical conditions that can be helped by a larger consumption of plant extracts as food supplements, which do not necessarily contain the specified antioxidant contents. By exploiting the catalytic properties of gold nanoparticles, a specific and sensitive nanoparticle-based label-free electrochemical sensor was developed, where the working parameters were optimized for RAC screening of hydrosoluble plant extracts. First, electrochemical measurements (cyclic voltammetry and amperometry) were used to characterize different nanoparticle-based sensors, revealing the best performance of gold nanoparticle-based sensors, obtaining a RAC of 98% for lavender extracts. The sensing principle is based on the quenching effect of antioxidants for H2O2 amperometric detection, where the decrease in electrical signal suggests an increasing antioxidant capacity. The obtained results were expressed in terms of ascorbic acid and Trolox equivalents in order to be able to correlate our results with classical methods like chemiluminescence and UV-Vis spectrophotometry, where a correlation coefficient of 0.907 was achieved, suggesting a good correlation between electrochemistry and spectrophotometry. Considering these results, the optimized gold nanoparticle-based label-free sensor can be used as a simple, rapid alternative towards classical methods for relative antioxidant capacity detection of hydrosoluble plant extracts.

Tyrosinase-Based Biosensors for Selective Dopamine Detection.

A novel tyrosinase-based biosensor was developed for the detection of dopamine (DA). For increased selectivity, gold electrodes were previously modified with cobalt (II)-porphyrin (CoP) film with electrocatalytic activity, to act both as an electrochemical mediator and an enzyme support, upon which the enzyme tyrosinase (Tyr) was cross-linked. Differential pulse voltammetry was used for electrochemical detection and the reduction current of dopamine-quinone was measured as a function of dopamine concentration. Our experiments demonstrated that the presence of CoP improves the selectivity of the electrode towards dopamine in the presence of ascorbic acid (AA), with a linear trend of concentration dependence in the range of 2-30 µM. By optimizing the conditioning parameters, a separation of 130 mV between the peak potentials for ascorbic acid AA and DA was obtained, allowing the selective detection of DA. The biosensor had a sensitivity of 1.22 ± 0.02 µA·cm-2·µM-1 and a detection limit of 0.43 µM. Biosensor performances were tested in the presence of dopamine medication, with satisfactory results in terms of recovery (96%), and relative standard deviation values below 5%. These results confirmed the applicability of the biosensors in real samples such as human urine and blood serum.

A new self-assembled layer-by-layer glucose biosensor based on chitosan biopolymer entrapped enzyme with nitrogen doped graphene.

The layer-by-layer (LbL) technique has been used for the construction of a new enzyme biosensor. Multilayer films containing glucose oxidase, GOx, and nitrogen-doped graphene (NG) dispersed in the biocompatible positively-charged polymer chitosan (chit(+)(NG+GOx)), together with the negatively charged polymer poly(styrene sulfonate), PSS(-), were assembled by alternately immersing a gold electrode substrate in chit(+)(NG+GOx) and PSS(-) solutions. Gravimetric monitoring during LbL assembly by an electrochemical quartz microbalance enabled investigation of the adsorption mechanism and deposited mass for each monolayer. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the LbL modified electrodes, in order to establish the contribution of each monolayer to the overall electrochemical properties of the biosensor. The importance of NG in the biosensor architecture was evaluated by undertaking a comparative study without NG in the chit layer. The GOx biosensor's analytical properties were evaluated by fixed potential chronoamperometry and compared with similar reported biosensors. The biosensor operates at a low potential of -0.2V vs., Ag/AgCl, exhibiting a high sensitivity of 10.5 µA cm(-2) mM(-1), and a detection limit of 64 µM. This study shows a simple approach in developing new biosensor architectures, combining the advantages of nitrogen-doped graphene with the LbL technique for enzyme immobilization.